Project Summary/Abstract To successfully repair the injured heart by rebuilding heart muscle rather than fibrotic scar is a fundamental goal of regenerative cardiovascular medicine. Toward this goal, targeting activated cardiac fibroblasts after injury to induce new cardiomyocytes is an attractive heart repair strategy. We and others have shown that the transduction of core cardiac transcription factors into fibroblasts is able to generate induced cardiomyocyte-like cells in vitro and in vivo (referred to as cardiomyocyte reprogramming). Over the last several years, significant progress has been made in cardiomyocyte reprogramming with markedly improved conversion efficiency in vitro. In contrast, cardiomyocyte reprogramming in situ for heart repair, which is an ultimate goal of this strategy, still remains a proof of concept approach without any further progress toward its clinical translation. Thus, there is a dire need to define the realistic therapeutic potential of this strategy for a post-myocardial infarction intervention. Toward this goal, we will re-program ?CM reprogramming approach? toward an effective post-MI intervention by addressing two fundamental pitfalls in its clinical translation. First, we will define the optimal combination of cardiac transcription factors, specifically tailored for CM reprogramming induced heart repair, rather than reprogramming cultured fibroblasts. Second, we will define the most effective therapeutic time window for cardiomyocyte reprogramming induced heart repair, which is clinically applicable, effective and safe. We envision that our re-programmed strategy executes cTFs induced heart repair with maximal efficacy and safety. Taken together, the results from this study will serve as a launching point for preclinical trials with large animals or non-human primates.